Project summary Necroptosis is a programmed form of necrotic cell death, which has been implicated in a variety of human diseases, including viral and bacterial infection, inflammation, ischemic tissue injuries as well as sepsis. Sepsis is the leading cause of mortality in critically ill patients and kills more than 258,000 people each year in the United States. The overall goal of this proposal is to dissect the molecular mechanisms of necroptosis, and develop novel necroptosis inhibitors that may help treat sepsis and other necroptosis-associated diseases. The defining molecular player for necroptosis is Receptor Interacting Protein Kinase 3 (RIP3), which binds its close homolog RIP1 upon activation. We have previously identified a small molecule inhibitor of necroptosis called necrosulfonamide and determined its target as Mixed lineage kinase domain-like protein (MLKL). Activated RIP1/RIP3 recruits MLKL to form necrosome. Phosphorylated MLKL then homo-oligomerizes and translocates to membrane compartments and triggers cell death. However, how MLKL membrane translocation is regulated and how cell death is executed is still not clear. We screened from 2,645 NCI compound collections and identified 12 hits that blocked necroptosis downstream of MLKL. Among them, compound NBC1 bound to HSP70 and compound NSC302979 is a known deubiquitinase (DUB) inhibitor. We will characterize the role of HSP70 and ubiquitination in regulating MLKL function and necroptosis. Both NBC1 and NSC302979 will also be tested in a mouse model of sepsis. We also conducted a CRISPR-Cas9 mediated whole genome knockout screen and identified list of genes required for necroptosis. Characterizing the role of these genes will lead to a better understanding of the pathway. Furthermore, using tandem immune-precipitation, we identified a MLKL interacting protein as Alix, which is involved in transporting ubiquitinated proteins to the lysosome. We also observed lysosome permeabilization during necroptosis. We hypothesize that Alix recruits ubiquitinated MLKL to the lysosome where MLKL complex permeabilizes lysosome and release lysosomal proteases and execute cell death. Involvement of lysosome permeabilization in necroptosis execution will be a significant conceptual advance in the field. In summary, our study uses a combination of chemical biology, biochemistry and genetics to decipher the mechanism of necroptosis. Completion of our aims will provide novel compound leads for treating sepsis and other necroptosis-associated disease, and will also identify new players in the pathway and provide better understanding of the underlying mechanisms.